Genetic and Functional Studies of the Intervertebral Disc: A Novel Murine Intervertebral Disc Model

Intervertebral disc (IVD) homeostasis is mediated through a combination of micro-environmental and biomechanical factors, all of which are subject to genetic influences. The aim of this study is to develop and characterize a genetically tractable, ex vivo organ culture model that can be used to further elucidate mechanisms of intervertebral disc disease. Specifically, we demonstrate that IVD disc explants (1) maintain their native phenotype in prolonged culture, (2) are responsive to exogenous stimuli, and (3) that relevant homeostatic regulatory mechanisms can be modulated through ex-vivo genetic recombination. We present a novel technique for isolation of murine IVD explants with demonstration of explant viability (CMFDA/propidium iodide staining), disc anatomy (H&E), maintenance of extracellular matrix (ECM) (Alcian Blue staining), and native expression profile (qRT-PCR) as well as ex vivo genetic recombination (mT/mG reporter mice; AdCre) following 14 days of culture in DMEM media containing 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin/streptomycin. IVD explants maintained their micro-anatomic integrity, ECM proteoglycan content, viability, and gene expression profile consistent with a homeostatic drive in culture. Treatment of genetically engineered explants with cre-expressing adenovirus efficaciously induced ex vivo genetic recombination in a variety of genetically engineered mouse models. Exogenous administration of IL-1ß and TGF-ß3 resulted in predicted catabolic and anabolic responses, respectively. Genetic recombination of TGFBR1^fl/fl explants resulted in constitutively active TGF-ß signaling that matched that of exogenously administered TGF-ß3. Our results illustrate the utility of the murine intervertebral disc explant to investigate mechanisms of intervertebral disc degeneration.     

The role of glyceraldehyde 3-phosphate dehydrogenase (GapA-1) in <Emphasis Type="Italic">Neisseria meningitidis</Emphasis> adherence to human cells

Background  

Glyceraldehyde 3-phosphate dehydrogenases (GAPDHs) are cytoplasmic glycolytic enzymes, which although lacking identifiable secretion signals, have also been found localized to the surface of several bacteria (and some eukaryotic organisms); where in some cases they have been shown to contribute to the colonization and invasion of host tissues. Neisseria meningitidis is an obligate human nasopharyngeal commensal which can cause life-threatening infections including septicaemia and meningitis. N. meningitidis has two genes, gapA-1 and gapA-2, encoding GAPDH enzymes. GapA-1 has previously been shown to be up-regulated on bacterial contact with host epithelial cells and is accessible to antibodies on the surface of capsule-permeabilized meningococcal cells. The aims of this study were: 1) to determine whether GapA-1 was expressed across different strains of N. meningitidis; 2) to determine whether GapA-1 surface accessibility to antibodies was dependant on the presence of capsule; 3) to determine whether GapA-1 can influence the interaction of meningococci and host cells, particularly in the key stages of adhesion and invasion.     

Morbid Jealousy and Sex Differences in Partner-Directed Violence

Previous research suggests that individuals diagnosed with morbid jealousy have jealousy mechanisms that are activated at lower thresholds than individuals with normal jealousy, but that these mechanisms produce behavior that is similar to individuals with normal jealousy. We extended previous research documenting these similarities by investigating sex differences in partner-directed violence committed by individuals diagnosed with morbid jealousy. The results support some of our predictions. For example, a greater percentage of men than women diagnosed with morbid jealousy used physical violence, attempted to kill, and actually killed their partners, and used their hands rather than an object to kill their partners. These results replicate results generated for individuals with normal jealousy. Discussion addresses implications of the current research and highlights directions for future research on the psychology of morbid jealousy.

Recombinant glycodelin carrying the same type of glycan structures as contraceptive glycodelin-A can be produced in human kidney 293 cells but not in chinese hamster ovary cells.

We have produced human recombinant glycodelin in human kidney 293 cells and in Chinese hamster ovary (CHO) cells. Structural analyses by lectin immunoassays and fast atom bombardment mass spectrometry showed that recombinant human glycodelin produced in CHO cells contains only typical CHO-type glycans and is devoid of any of the N, N'-diacetyllactosediamine (lacdiNAc)-based chains previously identified in glycodelin-A (GdA). By contrast, human kidney 293 cells produced recombinant glycodelin with the same type of carbohydrate structures as GdA. The presence of a beta1-->4-N-acetylgalactosaminyltransferase functioning in the synthesis of lacdiNAc-based glycans in human kidney 293 cells is concluded to be the cause of the occurrence of lacdiNAc-based glycans on glycodelin produced in these cells. Furthermore, human kidney 293 cells were found to be particularly suited for the production of recombinant glycodelin when they were cultured in high glucose media. Lowering the glucose concentration and the addition of glucosamine resulted in higher relative amounts of oligomannosidic-type glycans and complex glycans with truncated antennae. Human glycodelin is an attractive candidate for the development of a contraceptive agent, and this study gives valuable information for selecting the proper expression system and cell culture conditions for the production of a correctly glycosylated recombinant form.     

Autotomy of walking legs in the Pacific Shore crab Hemigrapsus oregonesis †

The ease of autotomy in Hemigrapsus oregonensis was tested by crushing the propodite of each walking leg. Individual curves of percent autotomy in groups of 18–21 animals were drawn for six different sequences of stimulation. Lumped data show peaks in ease of autotomy for the third anatomical leg of each side and for the second leg stimulated regardless of anatomical position. Later autotomies are more severely depressed when the stimulation sequence is from posterior to anterior. Segmental and lateral interaction and the characteristics of individual responses suggest that autotomy of legs in the crab, while depending upon specific structural features of the legs, is less a unisegmental reflex, than an accident occurring during escape.